Site-Directed Mutagenesis of Cytochrome P450eryF: Implications for Substrate Oxidation, Cooperativity, and Topology of the Active Site

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• 作者：Kishore K. Khan ; You Ai He ; You Qun He ; and James R. Halpert
• 刊名：Chemical Research in Toxicology
• 出版年：2002
• 出版时间：June 2002
• 年：2002
• 卷：15
• 期：6
• 页码：843 - 853
• 全文大小：187K
• 年卷期：v.15,no.6(June 2002)
• ISSN：1520-5010

文摘

The role of five active-site residues (Phe-78, Gly-91, Ser-171, Ile-174, and Leu-175) has beeninvestigated in P450eryF, the only bacterial P450 known to show cooperativity. The residueswere selected based on two-ligand-bound P450eryF structures and previous mutagenesis studiesof other cytochromes P450. To better understand the role of these residues in substrate catalysisand cooperativity, each mutant was generated in the wild-type and A245T background, asubstitution that enables P450eryF to oxidize testosterone and 7-benzyloxyquinoline (7-BQ).Replacement of Phe-78 with tryptophan decreased cooperativity of 9-aminophenanthrenebinding, with little effect on testosterone binding or oxidation. Interestingly, substitution ofGly-91 with alanine or phenylalanine abolished the type-I spectral change elicited bytestosterone and significantly decreased testosterone hydroxylation. However, G91A/A245Tshowed a 4-fold higher k_cat value with 7-BQ compared with A245T. Replacement of Ser-171with alanine or phenylalanine did not alter cooperativity of testosterone binding butsignificantly decreased binding affinity and oxidation of testosterone and 7-BQ. The only mutantthat exhibited an increased testosterone binding affinity and increased rates of testosteroneand 7-BQ oxidation was I174F. Substitution of Ile-175 with phenylalanine decreased testosterone and 7-BQ oxidation. Reaction with phenyldiazene showed that P450eryF may be muchmore open above pyrrole ring B than other cytochromes P450 and indicated significant changesin active-site topology in some of the mutants. The study suggests a crucial role of residuesSer-171, Ile-174, and Leu-175, which are part of a distal ligand site, in addition to the proximalGly-91 in determining the oxidative properties of P450eryF.